PROJECT SUMMARY Reducing the lower gastrointestinal toxicity of cancer chemotherapy is an unmet medical need. The dose- limiting toxicity of the anticancer drug irinotecan is intense diarrhea experienced at all grades by 88% of patients, and by 31% of patients at grades 3-4 that require significant medical attention. Irinotecan's active metabolite is inactivated by conjugation to a glucuronic acid moiety, which marks it for excretion into the intestines. The GI symbiotic microbiota, however, encode non-essential ?-glucuronidase (GUS) enzymes that scavenge the glucuronic acid sugar as a carbon source and release the reactivated antineoplastic compound into the intestinal lumen, where it kills epithelial cells and generates acute diarrhea. We have uniquely addressed this toxicity using microbiome-targeted inhibitors that selectively, potently, and non-lethally disrupt the activity of the microbial GUS enzymes to prevent drug reactivation. Here we propose to develop the most promising hits from our high-throughput screens into validated chemical probes with well understood structure activity relationships that alleviate irinotecan-induced GI toxicity. Our overarching hypothesis is that microbial enzymes expressed by the GI microbiome can be inhibited using targeted small molecules to prevent the unwanted reactivation of potent antineoplastic drugs in the intestinal lumen. We have assembled an interdisciplinary team of medicinal chemists, biochemists, structural biologists, and experts in animal models of chemotherapy all at the University of North Carolina at Chapel Hill. We will test this hypothesis using chemical synthesis, in vitro characterization, and in vivo validation in mouse models of chemotherapy-induced toxicity. This project will create the first microbiome-targeted compounds that rationally control a key mechanism of chemotherapy-induced toxicity. The probes generated will be made available to the field as research tools to broadly examine how modulating microbiome catalytic activities can affect chemotherapy, as well as other potential aspects of mammalian physiology.